中国免疫学杂志2011，Vol.27Issue(1)：15-19,5.DOI:10.3969/j.issn.1000-484X.2011.01.004
结核分枝杆菌Ag85A基因DNA疫苗的构建及其联合人IL-12表达质粒诱导的小鼠细胞免疫应答观察
Construction of DNA vaccine encoding of Ag85A gene of mycobacterium tuberculosis and observation on the immune responses in mice induced by co-immunization of Ag85A gene vaccine and plasmid encoding human interleukin 12
摘要
Abstract
Objective: To construct DNA vaccine based on Ag85A gene of mycobacterium tuberculosis, and to observe cellular immune responsees in mice induced by co-immunization of Ag85A gene vaccine and plasmid encoding human interleukin 12.Methods: ( 1 ) Constructing eukaryotic expressing plasmid. The gene encoding Ag85A mature protein was amplified by polymerase chain reaction ( PCR)from genome of mycobacterium tuberculosis H37Rv strain, which inserted into cloning vector PMD20-T after restriction endonuclease digestion. The gene fragment encoding Ag85A mature protein was correctly inserted into the vector, confirmed by restriction endonuclease digestion and partial nucleotide sequencing, and then was subcloned to the corresponding sites of eukaryotic expressing vector pcDNA3.1 ( + ). (2) Immunzation:50 C57BL/6N mice were randomly divided into following groups:① Ag85A gene vaccine plus plasmid of PORF- hlL-12;②Ag85A gene vaccine alone; ③ BCG( positive control); ④empty vector alone (negative control );⑤ PBS( blank group). The mice were immunized intramuscularly in both hind limbs 3 times at the intervals of three weeks or once subcutaneously with 1 × 106 of viable BCG (about 0.3 ml per mouse)at the first time of the DNA immunization. At the 28th days after the last immunization, the mice were killed,were splenocytes isolated. The splenocytes cytotoxicity-mediated was detected by LDH release assay,and proliferation of splenocyte was detected by XTT after stimulation of TB-PPD.The level of IFN-γ,IL-2 and IL-4 in the supernatant of spleno-lymphocyte cultures was measured by EILSA. Results: (1)The DNA vaccine entcoding Ag85A gene of mycobacterium tuberculosis was successfully constructed. (2)The co-immunization group could induce stronger Th1 cellular immune response. The levels of IFN-γ and IL-2 in supernatant of spleno-lymphocyte cultures in the co-immunization group were significantly higher than that of group of Ag85A alone,and they were similar to that in BCG group. The level of IL-4 decreased and was lower than that of BCG group.Specific spleen T cell cytotoxicity and splenocyte proliferation activities were significantly stronger than the other groups. Conclusion: Eukaryotic expressing plasmid PORF-hIL-12 can significantly reinforce the specific cellular immune responses in mice induced by the Ag85A DNA vaccine.关键词
结核分枝杆菌/Ag85A基因/DNA基因疫苗/人白细胞介素12质粒/联合免疫分类
医药卫生引用本文复制引用
丁珍珍,杜先智..结核分枝杆菌Ag85A基因DNA疫苗的构建及其联合人IL-12表达质粒诱导的小鼠细胞免疫应答观察[J].中国免疫学杂志,2011,27(1):15-19,5.基金项目
本文为国家自然科学基金资助项目(No.30872261) （No.30872261）
